Project Summary ? Biological Validation The validity of the Reference Interaction Map from Components 2 and 3, which rely primarily on ligation-based proximity mapping, will be assessed through independent methods testing genomic topology and its dynamics during cell cycle and cell differentiation. In addition, the correlations between topological features and other processes (e.g. local transcription rate, histone modifications, etc.) will imply functional roles for these features that need to be evaluated. We will use powerful imaging techniques to test and further elaborate the genomic structure and dynamics within our cellular systems. In addition, the direct perturbation of elements of specific topological features will define their biological roles in the cellular processes under study. To pursue these goals, we will develop a core set of tools and reagents and characterize a selected small number of TADs in depth. TADs from differentiating hESCs and from dividing fibroblasts will be selected for these analyses based on their dynamic topological behaviors and on the presence of embedded genes with dynamic expression patterns. By validating and perturbing topological features in both human ESCs and fibroblasts, we will be able to compare and contrast the similarities and differences in the behavior of these systems. These experiments should help define the basic grammar that underlies the formation, maintenance and dissolution of topological interactions. In Aim 1, we will establish clonal hESC and fibroblast ?imaging? lines, derived from the same lines that are being mapped and analyzed by the consortium, that harbor integrated imaging tools (e.g. nuclease-dead Cas9 [dCas9] variants tethered to fluorescent proteins). These cell lines will be used to (i) image dynamic TADs using multiple, independent methods to test whether their visible behavior is consistent with the structures and transitions inferred from sequencing approaches, and to (ii) measure the TAD-specific transcriptional consequences of dynamic topological behavior, as well as the topological consequences of dynamic transcriptional behavior. In Aim 2, we will engineer mutations in TAD boundaries and intra-TAD topological elements within these imaging cell lines, and use both imaging and molecular analyses to test the roles of the altered sequences in forming or maintaining genome topology. In Aim 3, we will use dCas9 variants fused to histone modification enzymes to define the topological and functional effects of ?writing? or ?erasing? specific chromatin marks within and around individual TADs. In addition we will probe the requirements for creating new topological features through the generation of artificial looping interactions via dCas9-interaction domains. The dataset generated through these studies will allow the more accurate parameterization of the computational models used to quantitatively represent and interpret the Reference Interaction Map as well as provide critical insights into the biological functions of the topological features contained within the map.